Nucleic acid molecules, RNA and DNA, present in both cellular and non-cellular samples are often associated with proteins or other macromolecules whose binding interferes with either detection or enzymatic manipulation of the nucleic acids. For this reason most protocols designed to detect or utilize nucleic acids begin with one or more purification and/or isolation steps that are carried out prior to any subsequent manipulation, such as amplification of particular target sequences or replication of reporter sequences.
Methods used to prepare the nucleic acid must be compatible with the subsequent biochemical steps. In addition, it is preferable to perform purification in the fewest reliable steps and the smallest possible number of containers in order to reduce losses. Quantitative accuracy and convenience of use are important features of assays, which may be carried out on large numbers of small samples, for instance samples composed of only one cell, or a relatively small number of cells, or samples comprised of a small piece of tissue, a fraction of a cell, or a small volume of a cellular extract or homogenate, or non-cellular samples of nucleic acids. Handling and processing, particularly of small samples, should not result in the loss, degradation, or contamination of said samples or of the nucleic acids within such samples.
Traditional methods for the purification or isolation of DNA and RNA from cells and tissues and the separation of DNA molecules from RNA molecules, typically include, but are not limited to: a) disruption/denaturation of the sample in the presence of strong denaturant agents such as guanidine salts, urea, detergents, strong alkali, or a combination of the above; b) separation of nucleic acids of interest from denatured proteins and/or other nucleic acids by extraction with a non-aqueous liquid, such as phenol:chloroform (used for total RNA and DNA separation), or by absorption to a matrix, resin, beads or fibers (used for selective extraction of mRNA and for other applications), or by neutralization with alkaline buffer and centrifugation (used for plasmid DNA isolation); c) recovery of nucleic acids by precipitation with an alcohol or a monovalent cation such as sodium or ammonium, or lithium chloride, and re-suspension to an appropriately small volume or, alternatively, elution of nucleic acids in an appropriately small volume. The volume in which the purified nucleic acids are contained determines the fraction of nucleic acids that can be analyzed in one assay. Volume is of particular importance when analysis is carried out on very small samples such as single cells or very few cells. It is known in the art that certain starting samples require the use of harsher conditions to disrupt cells. Conditions can be chosen to selectively degrade or digest DNA or RNA for recovery of the other. RNA molecules are much more sensitive to degradation than DNA molecules, due to their sensitivity to alkaline conditions, to high temperature, and to the ubiquitous presence of RNases. Thus, many protocols for isolation of RNA require milder conditions and include the presence of agents designed to inhibit RNases.
Examples of known methods for genomic DNA or RNA purification, isolation, or separation include use of: cetyltrimethylammonium bromide (CTAB) and high salt concentration, (Jones, A. S. (1963), Use of Alkyltrimethylammonium Bromides for the Isolation of Ribo- and Deoxyribo-Nucleic Acids, Nature 199:280-282); low salt concentration under hyperbaric, hydrostatic pressure (U.S. Pat. No. 6,111,096); gentle salt precipitation (and optional protease digestion); irreversible binding to aluminum oxide-covered matrixes (U.S. Pat. No. 6,291,166); preparation of RNA by guanidinium thiocyanate lysis followed by centrifugation through a CsCl cushion (Chirgwin, J. M. et al. (1979), Isolation of Biologically Active Ribonucleic Acid from Sources Enriched in Ribonuclease, Biochemistry 18:5294-5299); various modifications and improvements of the acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski, P. and Sacchi, N. (1987), Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction, Anal. Biochem. 162:156-159), later expanded to include DNA isolation (Chomczynski, P. (1993), A Reagent for the Single-Step Simultaneous Isolation of RNA, DNA and Proteins from Cell and Tissue Samples, Biotechniques 15:532-537); binding of total RNA or DNA to matrixes such as glass fiber filters, silica-gel membranes, magnetic beads or cellulose-based matrixes.
Specific extraction of mRNA molecules only is performed by interaction of their poly(A) tails with oligo (dT) attached to cellulose or resins containing a streptavidin complex; and selective precipitation of mRNA molecules with Poly T PNA probes and streptavidin. An intact poly(A) tail is probably not always present, particularly for very long RNA molecules such as Xist mRNA (Hong, Y. K. et al. (1999), A New Structure for the Murine Xist Gene and its Relationship to Chromosome Choice/Counting During X-Chromosome Inactivation, Proc. Natl. Acad. Sci. USA 96:6829-6834).
Fluorescence in situ hybridization (FISH) is an example of isolation and purification in situ. Procedures typically involve fixation of the sample, during which bound proteins are denatured, followed by washing steps in the presence of a detergent that removes at least some proteins. All these manipulations necessitate a considerable time-and-labor investment. Moreover, they all can lead to loss of nucleic acid molecules during the purification/extraction procedure or to poor detection of the target sequences. The latter can be a problem, since FISH probes need to recognize and visualize above background a very low number of target molecules.
Recently real-time target amplification methods, for example real-time PCR techniques, have provided a powerful tool for accurate quantification of RNA copy numbers, making possible the study of fine modulations of gene expression levels. As previously mentioned, however, RNA isolation is highly challenging, because of both the physical/chemical characteristic of this type of molecule and its sensitivity to the action of multiple RNases, present intracellularly or easily introduced by environmental contamination. Typically DNA is removed from RNA preparations by chemical, physical, or enzymatic methods. All of the above manipulations have contributed to shedding doubt on the reliability of RNA copy estimates obtained with the available protocols, particularly when analyzing very small samples. (See Klein, C. A. et al. (2002), Combined Transcriptome and Genome Analysis of Single Micrometastatic Cells, Nat. Biotechnol. 20:387-392).
Strategies have been devised to achieve mRNA capture, reverse transcription and PCR amplification in the same vessel, thereby limiting loss or damage of nucleic acids molecules during purification. Oligo (dT)-coated multi-well plates or streptavidin-coated PCR tubes to be used in conjunction with biotin-labeled oligo (dT)20, for example, are used for capture. Nucleic acids shearing may be lower with such methods, and loss of material due to transfer to a new vessel is avoided, but the accuracy of RNA quantification still depends on the efficiency of mRNA-binding to the capturing molecules.
Preparation of total RNA, rather than mRNA, is an alternative for the single-tube (or single-vessel, if microchips are used) approach, provided that removal of the proteins bound to nucleic acids is achieved in a way that doesn't interfere with subsequent steps of reverse transcription (RT) and PCR and does not affect RNA integrity.
Cell lysis by a simple freeze-thaw cycle neither separates proteins from nucleic acids, nor inactivates cellular RNases. Lysis by boiling bacterial cells surely leads to RNA hydrolysis.
A Cells-to-cDNA II Kit from Ambion, Inc. (Austin, Tex. U.S.A.) employs a Cell Lysis II Buffer compatible with RT and PCR. RNA copies can be thus amplified in one tube while DNA is degraded via DNase digestion, as suggested by the manufacturer. RT and PCR are then carried out sequentially by addition of the appropriate buffers and reagents to the lysed sample. The Cell Lysis II Buffer/RT PCR Buffer ratio tolerated by the assay, however, is low so that only a fraction of the lysed sample can be used for each RT-PCR assay. This technique, therefore, is not suitable for the analysis of very small samples, comprised of few or single cells. Non-ionic detergents, such as Triton® X-100 or NP40, are used in a number of protocols to lyse the cells plasma membrane and release cytoplasmic RNA pools. These detergents, at appropriate concentrations, are compatible with enzymatic reactions and cytoplasmic samples prepared with this method can be directly used in RT-PCR assays or other manipulation of RNA molecules aimed at their detection and/or quantification (Brady, G. and Iscove, N. N. (1993), Construction of cDNA Libraries from Single Cells, Methods Enzymol. 225:611-623; Hansis, C. et al. (2001), Analysis of October-4 Expression and Ploidy in Individual Human Blastomeres, Mol. Hum. Reprod. 7:155-161). Genomic DNA and nuclear RNA, such as Xist RNA, cannot, however, be prepared with these procedures.
Our laboratory developed an assay capable of measuring both genomic DNA and mRNA copies of genes of interest in single mouse embryos or blastomeres (Hartshorn, C., Rice J. E., Wangh, L. J. (2002), Developmentally-Regulated Changes of Xist RNA Levels in Single Preimplantation Mouse Embryos, as Revealed by Quantitative Real-Time PCR, Mol. Reprod. Dev. 61:425-436; Hartshorn, C., Rice, J. E., Wangh, L. J. (2003), Differential Pattern of Xist RNA Accumulation in Single Blastomeres Isolated from 8-cell Stage Mouse Embryos Following Laser Zona Drilling, Mol. Reprod. Dev. 64:41-51; Hartshorn, C., Rice, J. E., Wangh, L. J., Optimized Real-Time RT-PCR for Quantitative Measurements of DNA and RNA in Single Embryos and Blastomeres, In: Bustin S. A., ed. A-Z of Quantitative PCR, pages 675-702, International University Line, In press.). Counting genomic DNA copies in very small samples provides an ideal internal standard for nucleic acid recovery and for PCR efficiency. Moreover, there is no need to use DNase and the accompanying heat-inactivation of the enzyme, during which some RNA can be hydrolyzed (RNA degradation is enhanced by the presence of magnesium, required for DNase activity). Our attempts to adapt the Ambion Cells-to-cDNA II Kit to RT-PCR of RNA and DNA from single embryos/cells failed, probably due to the higher-than-recommended amount of Cell Lysis Buffer that had to be used in order to assay whole specimens rather than an aliquot of the specimen. While RNA was measured at the expected levels in high-expressing samples, genomic DNA, present in low copy numbers, was very often undetected.
Examples of lysis buffers aimed at the preparation of DNA templates only and compatible with PCR analysis in the same reaction vessel are known and commercially available. Generally these methods do not allow simultaneous/parallel analysis of DNA and RNA molecules from the same samples, because RNA is degraded during the extraction procedure. The Release-IT™ (CPG, Inc., Lincoln Park, N.J., U.S.A.) is a proprietary reagent that releases DNA from whole blood, cell cultures, bacterial colonies and other biological samples. Lysis is accomplished directly in the amplification tube on a thermal cycler, and PCR reagents are subsequently added to the lysate initiating amplification. Release-IT™ sequesters cell lysis products that might inhibit PCR. Unlike other methods described above, this allows RNA recovery and reverse transcription of small aliquots of the sample for RT-PCR (the whole sample can be used for PCR of genomic DNA). However, the initial heating cycle required for Release-IT™ action is believed to be detrimental to RNA, because it includes a total of 4 minutes at 97° C. and a holding step at 80° C. Moreover, the Release-IT™ reagent is not recommended for amplification of low copy number DNA without cellular enrichment.
Nucleic acids analysis in very small samples, including single cells or a portion of a single cell, presents a number of challenges. While several commercial kits offer RT-PCR sensitivity down to the single-cell level, this claim often implies harvesting a larger sample of which only a portion is used for each assay. A few kits promise efficient nucleic acids extraction from actual single cells, but collection of the individual samples themselves is frequently difficult and should be done carefully to preserve RNA content (Hartshorn, C., Rice, J. E., Wangh, L. J. (2003)). Recently, laser capture microdissection (LCM) and laser pressure catapulting (LPC) have made precise excision of single cells, or compartments of single cells, possible. Further, two techniques have already been developed for single-cell gene expression profiling that rely on polyadenylation of mRNA molecules for their direct detection in a cell lysate, without need for RNA purification (3 prime end amplification, or TPEA, and global amplification of cDNA copies of all polyadenylated mRNAs, or PolyAPCR) (reviewed by Brady, G. (2000), Expression Profiling of Single Mammalian Cells—Small is Beautiful, Yeast 17:211-217). Both techniques are limited to cytoplasmic RNA measurements and do not extend to DNA or nuclear RNA.
An aspect of this invention is a method for preparing DNA or RNA molecules, or both, for amplification and detection or for other enzymatic processing of mixtures of DNA and RNA molecules that have been freed of bound proteins, such mixtures comprising freed DNA and RNA molecules, a chaotropic agent, and degraded and denatured proteins, comprising diluting the mixture so as to reduce the concentration of chaotropic agent to less than 0.05 M, preferably less than 0.01 M, without removing or isolating the DNA and RNA molecules from each other or from the other components of the mixture.
Another aspect of this invention is preparing the foregoing mixture, prior to diluting it, by incubating a sample containing protein-bound DNA and RNA molecules with a concentrated disruption reagent containing at least 2 M chaotropic agent.
Another aspect of this invention is amplifying one or more DNA and RNA sequences in the diluted mixture without physically separating the DNA and RNA molecules from the diluted mixture.
A further aspect of this invention is performing the foregoing dilutions and amplifications in a single container and, preferably, also preparing the mixture in the container.
Yet another aspect of this invention is a device useful in freeing DNA and RNA molecules from bound proteins comprising a dry or semi-dry disruption reagent containing a chaotropic reagent that is adhered to a surface of a container or a container part, such as a cap of a tube or a surface within a multiple-chambered container.
Another aspect of this invention is kits including the foregoing device and additional reagents and materials for performing dilution and further processing of diluted mixtures, for example, amplification and sequencing reactions.